DESCRIPTION: (summarized from abstract) Chemokines are small molecular weight inflammatory mediators responsible for chemoattracting neutrophils, monocytes and lymphocytes into sites of inflammation. The PI has discovered that IL-1a stimulated human corneal epithelial cells (HCEC) synthesize the a-chemokine IL-8 but not the b-chemokine MCP-1. The goal for this project is to determine if combinatorial interaction between NF-kB and C/EBP transcriptional factors accounts for the differential expression of the two chemokines in HCEC and to determine how the amounts of IL-8 produced by HCEC is regulated. RT-PCR, immunoblotting and electrophoretic mobility shift assays will be used to determine which members of the NF-kB and C/EBP family of transcriptional activators are expressed and activated in HCEC. To determine if transcriptional factors activated in IL-1a stimulated cells stimulate IL-8 transcription without stimulating MCP-1 transcription, reporter plasmids will be constructed in which NF-kB and C/EBP binding elements of the IL-8 and MCP-1 promoters are used to drive expression of the luciferase gene. The expression of reporter plasmids transfected into HCEC will then be monitored following IL-1a stimulation. RT-PCR and ELISA assays will be used to test the hypothesis that the same combination of transcriptional factors that activate IL-8 expression will also activate expression of ENA-78, a a-chemokine whose promoter possesses both sequence homology and structure similarity to the IL-8 promoter. The role of IL-1 receptor antagonist (IL-1RA) and soluble IL-1 type 2 receptor (IL-1RII) proteins in dampening HCEC responses to IL-1a will be tested by neutralizing these naturally produced epithelial cell inhibitors with anti-IL-1RA, and anti-IL-1RII antibodies during IL-1a stimulation. This study will increase our understanding of the molecular events which precipitate inflammatory responses on the eye surface.